Method for optical DNA mapping based on scanning electron microscopy (SEM)

Abstract

A method of optical DNA mapping based on scanning electron microscopy (SEM) is provided. The method of optical DNA mapping according to one aspect provides enhanced accessibility and user friendliness, excellent compatibility with chemically functionalized surfaces and microfluidic devices, and the ability to obtain high resolution DNA images without the use of metals, by utilizing SEM as a base platform, as well as to provide a high resolution DNA imaging system. Therefore, optical DNA mapping with improved resolution can be realized.

Description

[0001] Cross-reference to related applications

[0002] This application is based on and claims the benefit of priority under 35 U.S.C. §119 from Korean Patent Application No. 10-2024-0036772 filed on March 15, 2024, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference. Technical Field

[0003] The present disclosure relates to a method for scanning electron microscopy (SEM)-based optical DNA mapping.

[0004] The research described in this article was performed with support from the Samsung Future Technology Cultivation Project (Project No. SRFC-MA2101-07). Background Art

[0005] Optical DNA mapping is a technology for constructing an orderly whole genome map from a single dyed DNA molecule. Single molecule DNA sequencing technology is developing rapidly, but usually relies on reading genomes in fragments less than 1000bp. The main purpose of optical DNA mapping is to supplement DNA sequence analysis by providing a large-scale scaffold, and these short sequence reads can be assembled on the scaffold. Specifically, optical DNA mapping can guide the assembly of complex genomic regions when combined with a base sequencing method. This makes it a useful tool for reaching the DNA length scale that conventional sequencing cannot reach. This optical DNA mapping requires an imaging technology that can visualize DNA molecules with high effectiveness.

[0006] The method for DNA analysis and observation in the related art includes microscopic techniques, such as transmission electron microscopy (TEM), fluorescence microscopy (FM) and scanning electron microscopy (SEM). Although transmission electron microscopy (TEM) can observe the fine structure of DNA in detail, it needs to carry out DNA imaging to overcome low electron scattering by using heavy metal salts (such as uranyl acetate) in combination with shadow projection method. For this reason, a large amount of time and complicated process are needed to observe DNA molecules by TEM. Fluorescence microscopy (FM) is widely used due to its simplicity, accessibility and real-time imaging ability, but the inherent limitation of its low resolution makes it challenging to accurately observe DNA molecules on nanoscale. One of the key requirements of DNA imaging is the compatibility with microfluidic devices and chemical functionalized surfaces. Although the DNA images obtained by FM typically show good alignment and parallel extension, TEM faces difficulties in realizing this type of alignment due to the alignment problem of the metal grid coated with carbon film, resulting in DNA molecules that are usually randomly arranged. SEM can achieve both nanometer resolution and millimeter-level observation range. Furthermore, it offers greater flexibility in accommodating functionalized surfaces and microfluidic devices for DNA molecule alignment. Therefore, SEM is considered a method that can address the shortcomings of both transmission electron microscopy and fluorescence microscopy. However, SEM is limited in that its resolution far exceeds the 2 nm thickness of DNA. Various methods have been developed to enhance the visibility of DNA molecules, including nanoparticle attachment and nanowire growth. However, these methods have primarily been used to visualize artificially constructed DNA nanostructures and encounter significant challenges when applied to genomic DNA.

[0007] Therefore, there is a need for a new optical DNA mapping technique that can achieve high-resolution images comparable to TEM while retaining many of the advantages of FM, such as simplicity, accessibility, and the ability to obtain well-aligned DNA images. Summary of the Invention

[0008] One aspect is to provide a method for optical DNA mapping based on scanning electron microscopy (SEM), comprising: (a) contacting double-stranded DNA with a nicking enzyme to form a nick at a target sequence motif; (b) generating labeled double-stranded DNA by incorporating labeled nucleotides into the nicking site of the double-stranded DNA; (c) staining the backbone of the labeled double-stranded DNA; (d) stretching and immobilizing the stained double-stranded DNA on a substrate having a functionalized surface; and (e) determining the distance between each marker by imaging the immobilized double-stranded DNA using a scanning electron microscope (SEM).

[0009] Additional aspects will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the presented embodiments of the disclosure.

[0010] One aspect provides a method for optical DNA mapping based on scanning electron microscopy (SEM), comprising: (a) contacting double-stranded DNA with a nicking enzyme to form a nick at a target sequence motif; (b) generating labeled double-stranded DNA by incorporating labeled nucleotides into the nicking site of the double-stranded DNA; (c) staining the backbone of the labeled double-stranded DNA; (d) stretching and immobilizing the stained double-stranded DNA on a substrate having a functionalized surface; and (e) determining the distance between each marker by imaging the immobilized double-stranded DNA using a scanning electron microscope (SEM).

[0011] The term "optical DNA mapping" refers to a technique that uses imaging technology to provide positional information covering the entire genome from a single DNA molecule. Such positional information can be combined with sequence analysis data to serve as a guide for genome sequence assembly.

[0012] The term "scanning electron microscope (SEM)" refers to a device that performs microstructural observation by scanning a sample with an electron beam, detecting signals emitted from the sample surface with a detector, and displaying their intensity as a three-dimensional image on a monitor to obtain a magnified image of the sample. Scanning electron microscopes may include thermionic emission SEMs or field emission SEMs.

[0013] A step-by-step explanation of the method of SEM-based optical DNA mapping is provided below.

[0014] First, the method can include (a) contacting double-stranded DNA with a nicking enzyme to form a nick at a target sequence motif; and (b) generating labeled double-stranded DNA by incorporating labeled nucleotides into the nicking site of the double-stranded DNA.

[0015] Steps (a) and (b) are steps for sequence-specific labeling of DNA molecules by nick translation.

[0016] The term "nickase" refers to an endonuclease that cleaves only one strand of double-stranded DNA, ie, introduces a nick into the double-stranded DNA.

[0017] Nicking enzyme can be a nicking endonuclease, and term " nicking endonuclease (nickingendonuclease) " refers to the endonuclease that recognizes target sequence motif to form sequence-specific nick.Nicking endonuclease can be naturally occurring, or can be engineered by restriction endonuclease.In one embodiment, nicking endonuclease can be Nb.BbvCI, Nb.BssSI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI or Nt.CviPII, but is not limited to this.Any endonuclease that can recognize target sequence motif and form nick in double-stranded DNA molecule can be used according to the method for the present disclosure.More than 200 kinds of nicking endonucleases have been studied, and some of these can be obtained from the source business such as New England Biolabs. The target sequence motif can be a short nucleotide sequence of 8 or fewer bases. For example, Nb.BbvCI recognizes and cuts the 5'-CC^TCAGC-3' site, and Nb.BssSI recognizes and cuts the 5'-C^TCGTG-3' site.

[0018] Alternatively, the nickase can be an RNA-guided endonuclease, and the term "RNA-guided endonuclease" refers to an endonuclease that recognizes a target sequence motif by a guide RNA (gRNA) and forms a sequence-specific nick by forming a complex with the guide RNA. The RNA-guided endonuclease can be, for example, nCAS9 (cas9 nickase), and when the nickase is an RNA-guided endonuclease, the nickase can be used with a guide RNA sequence that includes a portion of a sequence complementary to the target double-stranded DNA.

[0019] In one embodiment, the nicking enzyme can be Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, nCAS9, or a combination thereof.

[0020] The term "nick" refers to a site of broken phosphodiester bonds in double-stranded DNA, and the introduction of a nick can expose free 3' and free 5' strands at that site. That is, the nick can include a 3' hydroxyl group and an adjacent 5' phosphate group.

[0021] The term "nick translation" refers to the process by which a polymerase extends a DNA strand from the 3' hydroxyl group at the nick site while simultaneously degrading the adjacent strand.

[0022] In one embodiment, the labeled nucleotide can be a nucleotide that is conjugated directly or indirectly to a detectable label.The detectable label can be conjugated to the nucleotide by covalent bonding or by a binding pair of a first binding moiety and a second binding moiety.

[0023] The term "detectable label" refers to a label that can be detected by optical methods, i.e., an optical label. A detectable label can be a label that is detectable under SEM, and can be a label that can be brightly visualized in contrast to the DNA backbone that appears as a dark line under SEM by a staining step.

[0024] In one embodiment, the detectable label can be a nanoparticle or a fluorescent dye. The nanoparticle can be any one selected from the group consisting of metal nanoparticles, oxide nanoparticles, sulfide nanoparticles, nanoclusters (nanocluster), quantum dots, graphene quantum dots, perovskite (perovskite), carbon dots, polymer particles, hydroxyapatite (hydroxyapatite) and magnetic nanoparticles. The metal nanoparticle can be, for example, a gold nanoparticle. Fluorescent dyes can be, for example, cyanine dyes (e.g., Cy3, Cy5, and Cy7 dyes); xanthenedyes (e.g., fluorescein isothiocyanate (FITC), 6-carboxyfluorescein, 6-carboxy-2',4',7,4,7-hexachlorofluorescein (HEX), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine 6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110 (rhodamine 110); 110) and other fluorescein and rhodamine dyes); and benzimide dyes (e.g., Hoechst 33258); phenanthridine dyes (e.g., Texas Red). The diameter of the nanoparticles can be 5 nm to 20 nm. When the diameter of the nanoparticles exceeds the aforementioned range, introduction into the incision site may be difficult, and when the diameter is below the aforementioned range, observation under SEM may be difficult.

[0025] In one embodiment, step (b) may comprise: contacting the nicked double-stranded DNA with a DNA polymerase in the presence of at least one nucleotide conjugated to a first binding portion to generate a modified double-stranded DNA having the first binding portion; and contacting the modified double-stranded DNA with a detectable label conjugated to a second binding portion to generate a labeled double-stranded DNA, wherein the second binding portion forms a binding pair with the first binding portion.

[0026] In one embodiment, the first binding moiety or the second binding moiety can be any one selected from the group consisting of biotin, streptavidin, digoxin, neutravidin, and avidin. For example, the first binding moiety and the second binding moiety can form a binding pair of biotin / streptavidin, biotin / neutravidin, or biotin / avidin.

[0027] In one embodiment, the nucleotide conjugated to the first binding moiety can be biotin-dUTP, and the detectable label conjugated to the second binding moiety can be a detectable label coated with streptavidin.

[0028] In one embodiment, the DNA polymerase can have 5'→3' exonuclease activity. The DNA polymerase can remove original nucleotides in the 5'→3' direction and simultaneously replace them with labeled nucleotides through its 5'→3' polymerase activity. The DNA polymerase can be DNA polymerase I.

[0029] The step of generating modified double-stranded DNA can include contacting the double-stranded DNA with one or more nucleotides conjugated to a first binding moiety, one or more nucleotides (dNTP-dTTP, dATP, dCTP, and dGTP), a DNA polymerase, or a ligase.

[0030] Furthermore, the method may include (c) staining the backbone of the labeled double-stranded DNA.

[0031] This step is for electrostaining DNA for visualization under a scanning electron microscope (SEM).

[0032] Figure 1 is a schematic diagram illustrating DNA electrostaining using a DNA-binding protein and a polymer according to an embodiment.

[0033] According to one aspect, the staining reagent used in the method can be prepared by combining a DNA binding protein with a synthetic polymer, and referring to Figure 1, DNA can be stained by forming a complex with DNA-binding proteins and polymers that can bind to DNA.

[0034] In one embodiment, step (c) may comprise contacting the labeled double-stranded DNA with a DNA binding protein.

[0035] DNA binding proteins can bind to DNA molecules through one or more of the following interactions: electrostatic interaction, intercalation, or groove binding. For example, the peptide in the embodiment can include positively charged amino acid residues that electrostatically interact with negatively charged DNA molecules. The peptide that binds to the DNA molecule through electrostatic interaction can include an amino acid sequence containing at least one selected from arginine, histidine, and lysine. Groove binding refers to the interaction between the exposed bases on the DNA molecule and the peptide comprising the amino acid sequence, so that the peptide can bind to the DNA through groove binding. Intercalation is the phenomenon that molecules, atoms, or ions are inserted between the layers of a layered material; therefore, a peptide comprising the above-mentioned amino acid sequence can be inserted into a DNA molecule. In such a case, the peptide that binds to the DNA molecule through intercalation can include an amino acid sequence containing at least one aromatic amino acid ring selected from phenylalanine, tyrosine, and tryptophan.

[0036] In addition to electrostatic interactions, the peptide may undergo groove binding or intercalation with the DNA molecule, or may undergo both intercalation and groove binding as well as electrostatic interactions.

[0037] The peptide may have one or more functional groups at at least one of its N-terminus or C-terminus. Such functional groups may include one or more selected from thiol groups, amine groups, carboxyl groups, cysteine ​​groups, azide groups, alkyne groups, glutaraldehyde groups, and maleimide groups, but are not limited thereto, and may encompass all peptides that can be used for DNA binding proteins. For example, the peptide may be bound to a DNA molecule, thereby providing the DNA molecule with the above-mentioned functional groups.

[0038] In one embodiment, the DNA binding protein may include a truncated transcription activator-like effector (tTALE), a histone-like nucleoid-structural protein (H-NS), Cro or a high mobility group (HMG) protein, etc.

[0039] In one embodiment, the DNA binding protein can further include a fluorescent protein. DNA binding proteins such as H-NS or fluorescent proteins can serve as excellent docking sites for polymers described below. In addition, the oligomerization of fluorescent proteins can enhance the binding strength with polymers described below and can exist as an assembly around the DNA molecule together with polymers described below.

[0040] In one embodiment, the fluorescent protein may include mScarlet, EmGFP, mNeonGreen, RRvT, tdTomato, mClover3, YPet, mOrange2, Emerald, mTurquoise2, mStrawberry, and mCherry, among others.

[0041] In one embodiment, the DNA binding protein can be connected to a fluorescent protein through a joint, or the DNA binding protein (DNA-binding protein, DBP) and fluorescent protein (fluorescent protein, FP) can be directly connected without a joint. Examples of such DNA binding protein-fluorescent proteins can include truncated TALE (tTALE)-emGFP, H-NS-mScarlet, Cro-mNeonGreen and mNeonGreen-HMG, etc. Such DNA binding protein-fluorescent proteins can affect the brightness required for visualizing DNA molecules by electron microscopy and the extension of DNA molecules.

[0042] In one embodiment, step (c) may further comprise contacting the double-stranded DNA with a polymer capable of binding to a DNA-binding protein, an anhydrate thereof, or a salt thereof.

[0043] In addition to the polymer itself, the polymer capable of binding to the DNA binding protein may also include an anhydrate or a salt thereof. The salt may be an inorganic salt or an organic salt. For example, the salt may be a cationic inorganic salt.

[0044] The polymer may be able to bind to the DNA binding protein through intermolecular electrostatic attraction.

[0045] According to one embodiment, the polymer may include one or more oxygen atoms and nitrogen atoms.

[0046] According to one embodiment, the oxygen atoms or nitrogen atoms of the polymer can be bonded to the hydrogen atoms of the OH bonds or NH bonds contained in the DNA binding protein through hydrogen bonds. This hydrogen bond corresponds to intermolecular bonding and is different from chemical bonding.

[0047] According to one embodiment, the polymer, its anhydrate or its salt may be in the form of aggregates.

[0048] In one embodiment, the polymer may include one or more structural units represented by the following formulae 1 to 9:

[0049] [Formula 1] [Formula 2]

[0050] , ,

[0051] [Formula 3] [Formula 4] [Formula 5]

[0052] , , ,

[0053] [Formula 6] [Formula 7]

[0054] , ,

[0055] [Formula 8]

[0056] ,

[0057] [Formula 9]

[0058]

[0059] In formulas 1 to 9,

[0060] m+n can be equal to 1.

[0061] In one embodiment, the polymer, its anhydrate, or its salt may have a weight-average molecular weight (Mw) of 10 kDa to 100 kDa, for example, 20 kDa to 100 kDa, 30 kDa to 100 kDa, 40 kDa to 100 kDa, 10 kDa to 90 kDa, 20 kDa to 90 kDa, 30 kDa to 90 kDa, 40 kDa to 90 kDa, 10 kDa to 60 kDa, 20 kDa to 60 kDa, 30 kDa to 60 kDa, 40 kDa to 60 kDa, or 30 kDa to 50 kDa.

[0062] According to one embodiment, when used in conjunction with the above-described DNA-binding protein, the polymer, its anhydrate, or its salt can stain DNA molecules black with an average thickness of approximately 15.0±4.0 nm, thereby facilitating visualization of the DNA molecules by SEM. As demonstrated in one embodiment, this method enables high-resolution imaging of not only linear, circular, single-stranded, or double-stranded DNA configurations, but also a wide range of DNA structures, from genomic and mitochondrial DNA to chromatin and chromosomes.

[0063] Because the method according to one aspect can enhance the visibility of 2nm thick DNA molecules for SEM imaging without using heavy metals such as uranyl acetate, which are conventionally used as electron microscopy staining reagents, step (c) can be a metal-free staining step. Therefore, unlike transmission electron microscopy (TEM), this method does not require a lot of time or complicated procedures to observe DNA molecules. In addition, in contrast to most electrical staining techniques, the method according to one aspect is stained without the use of metals, resulting in DNA appearing as continuous dark lines under SEM. As a result, high-resolution SEM images of various DNA forms, including linear, circular, double-stranded, single-stranded, and even RNA, can be obtained, and human cell genomes and mitochondrial DNA molecules, as well as transcribed chromatin and chromosomes, can be visualized.

[0064] In addition, the method may further include (d) stretching and immobilizing the dyed double-stranded DNA on a substrate having a functionalized surface.

[0065] In one embodiment, the substrate having a functionalized surface can be a silicon substrate that has been surface-modified to carry a positive charge. The silicon substrate can be a silicon substrate having a SiO2 layer formed thereon. The surface modification can be performed with an ammonium salt. The silicon substrate can be advantageously used to easily accommodate a PDMS microfluidic device.

[0066] Positively charged surface-modified silicon substrates are used to immobilize DNA molecules. In contrast, the glass substrate surface typically used for fluorescence microscopy (FM) induces an electron charging effect and is therefore unsuitable for observing DNA molecules by SEM. In addition, substrates coated with conductive metals such as gold (Au) or platinum (Pt) blur the 2nm thick DNA image under SEM, making such substrates unsuitable for DNA observation. The silicon substrate used in this disclosure is capable of achieving effective charge transfer due to its semiconductor properties.

[0067] A positively charged surface-modified silicon substrate can be prepared by contacting an ammonium salt precursor with a silicon substrate having a SiO2 layer formed thereon. The silicon substrate having the SiO2 layer formed thereon and modified with the positive charge from the ammonium salt is used to immobilize the DNA backbone. The thickness of the SiO2 layer can range from a few nanometers to tens of nanometers. The thickness of the SiO2 layer can be appropriately adjusted within a range that maintains the positive charge after surface modification with positive charge.

[0068] The ammonium salt precursor may include one or more selected from the group consisting of ammonium hydroxide, ammonium chloride compounds, ammonium sulfate compounds, ammonium carbonate compounds, ammonium bicarbonate compounds, and ammonium acetate compounds. For example, the ammonium salt precursor may be an ammonium chloride compound. For example, the ammonium salt precursor may be N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride.

[0069] In an embodiment, step (d) may include passing the stained double-stranded DNA through a microchannel. Various nucleic acid stretching methods known in the related art can be used for linear stretching of DNA. According to one embodiment, the microfluidic device can provide uniform and efficient DNA stretching through a microchannel, thereby producing well-aligned parallel images when observed by a scanning electron microscope (SEM). DNA stretching through a microchannel can be used to maximize the information available from optical DNA mapping. The microchannel can be a polydimethylsiloxane (PDMS) microchannel, which is used to guide a DNA solution (which has been labeled and stained) onto a silicon substrate surface. In an embodiment, step (d) may further include drying before removing the microchannel from the substrate. For example, the DNA loaded on the silicon substrate surface can be dried at room temperature for about 30 minutes to 1 hour to fully immobilize the DNA, which can then be imaged by SEM.

[0070] Because the patterning method according to one aspect employs SEM as the basic platform, the method can be seamlessly integrated with chemically functionalized surfaces and microfluidic devices.

[0071] Additionally, the method may include (e) determining the distance between each marker by imaging the immobilized double-stranded DNA using a SEM.

[0072] This step involves detecting the labeling pattern on the stretched double-stranded DNA by SEM imaging, thereby generating positional information. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The above and other aspects, features and advantages of certain embodiments of the present disclosure will become more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0074] Figure 1 is a schematic diagram illustrating DNA electrostaining using a DNA binding protein and a polymer according to one embodiment;

[0075] Figure 2 is a schematic diagram of a PDMS microfluidic device according to one embodiment;

[0076] Figure 3 is an SEM image of a bacteriophage λ DNA molecule obtained by stretching / immobilization and imaging in the same manner as Example 1-1 without any staining agent treatment (untreated control);

[0077] Figure 4 Shown are SEM images of bacteriophage lambda DNA molecules stained with mNG-HMG alone, then stretched / immobilized and imaged (Comparative Example 1-1), and corresponding FM images;

[0078] Figure 5 is an SEM image of a bacteriophage lambda DNA molecule stained with PVP alone, then stretched / immobilized and imaged (Comparative Examples 1-2);

[0079] Figure 6 is an SEM image of a bacteriophage lambda DNA molecule stained with a combination of H-NS and PVP, then stretched / immobilized and imaged (Examples 1-2);

[0080] Figure 7 SEM images of bacteriophage lambda DNA molecules stained with a combination of H-NS-mScarlet and PVP, then stretched / immobilized and imaged (Examples 1-3) are shown, and corresponding FM images are also shown;

[0081] Figure 8 SEM images of bacteriophage lambda DNA molecules stained with a combination of tTALE-EmGFP and PVP, then stretched / immobilized and imaged (Examples 1-4), and corresponding FM images are shown;

[0082] Figure 9SEM images of bacteriophage lambda DNA molecules stained with a combination of Cro-mNeonGreen and PVP, then stretched / immobilized and imaged (Examples 1-5), and corresponding FM images are shown;

[0083] Figure 10 SEM images of bacteriophage lambda DNA molecules stained with a combination of mNG-HMG and poly(2-ethyl-2-oxazoline) followed by stretching / immobilization and imaging are shown (Examples 1-6), and SEM images of bacteriophage lambda DNA molecules stained with the same combination and immobilized on a positively charged surface-modified silicon wafer without the use of a microfluidic device and imaged are also shown;

[0084] Figure 11 SEM images of bacteriophage lambda DNA molecules stained using a combination of mNG-HMG and polyaniline, then stretched / immobilized and imaged are shown (Examples 1-7);

[0085] Figure 12 SEM images of bacteriophage lambda DNA molecules stained with polyaniline alone and then immobilized on a positively charged surface-modified silicon wafer without using a microfluidic device are shown (Comparative Examples 1-3);

[0086] Figure 13 shows SEM visualization of PVP droplets on various functionalized silicon wafer surfaces, namely, a positively charged surface-modified silicon wafer, a silicon wafer with a SiO2 layer, a flat silicon wafer, and a platinum-coated silicon wafer, wherein the dotted line in the platinum-coated wafer image indicates the boundary of the PVP droplet;

[0087] Figure 14A and 14B The SEM imaging results and the corresponding FM imaging results of bacteriophage λ DNA molecules stained with a combination of mNG-HMG and PVP and then applied and immobilized on the surface of a positively charged surface-modified silicon wafer and imaged without using a microfluidic device are shown, respectively;

[0088] Figure 15 Shows the SEM imaging results of bacteriophage lambda DNA molecules stained with a combination of mNG-HMG and PVP, then stretched / immobilized and imaged (Example 1-1);

[0089] Figure 16AThe SEM imaging results of a bacteriophage λ DNA (48.5 kb) molecule stained with a combination of mNG-HMG and PVP, then stretched / immobilized and imaged (Example 1-1), and the corresponding FM imaging results are shown, and Figure 16B Shown from Figure 16A An inverted magnified version of the SEM image of bacteriophage λ DNA (48.5 kb), including the intensity distribution and the corresponding full width at half maximum (FWHM) measured along the yellow line of the inverted SEM DNA image;

[0090] Figure 16C Shown are the SEM imaging results of circular M13mp18 double-stranded DNA (7.2 kb) molecules stained with a combination of mNG-HMG and PVP, then stretched / immobilized and imaged (Example 2-1), and the corresponding FM imaging results;

[0091] Figure 17A and 17B The SEM imaging results (Example 2-2) and the corresponding FM imaging results of genomic DNA molecules extracted from HeLa cells, stained using a combination of mNG-HMG and PVP, and then stretched / immobilized and imaged, are shown, respectively, and Figure 17C shows SEM imaging results illustrating Christmas tree-like DNA structures potentially representing transcription along the backbone of genomic DNA derived from HeLa cells;

[0092] Figure 18A and 18B The SEM imaging results (Example 2-2) and the corresponding FM imaging results of mitochondrial DNA (16.6 kb) extracted from HeLa cells, stained with a combination of mNG-HMG and PVP, and then stretched / fixed and imaged are shown respectively;

[0093] Figure 19A The figure shows the results of FM imaging after metaphase chromosomes extracted from HeLa cells were immobilized on a positively charged surface-modified silicon wafer and stained with YOYO-1 staining reagent;

[0094] Figure 19B The figure shows the results of SEM imaging after metaphase chromosomes extracted from HeLa cells were immobilized on a positively charged surface-modified silicon wafer and then stained with PVP only without DNA-binding protein;

[0095] Figure 19Cshows the results of SEM imaging after immobilizing metaphase chromosomes extracted from HeLa cells on a positively charged surface-modified silicon wafer without staining with any DNA-binding protein or PVP, thereby observing the chromosomes in their native state;

[0096] Figure 20A is a schematic diagram illustrating DNA stained with mNG-HMG (DBP-FP) and PVP (shown as black circles) and labeled with streptavidin-coated quantum dots, and Figure 20B and 20C An enlarged view of the QD-labeled site from the SEM-based optical map of the bacteriophage λ DNA generated in Example 3-2 and an enlarged view of the QD-labeled site from the corresponding FM-based optical map are respectively shown;

[0097] Figure 21A is a schematic diagram illustrating that circular M13mp18 double-stranded DNA (7.2 kb) contains two nicking sites at 1373 bp and 1417 bp and that DNA polymerase travels in two opposite directions, and Figure 21B and 21C The results of observing QD labels in the SEM-based optical map of the circular M13mp18 double-stranded DNA generated in Example 3-1 and the corresponding QD labels observed in the FM-based optical map are respectively shown;

[0098] Figure 22 The SEM-based DNA optical mapping results and the corresponding FM-based optical mapping results of the phage λ DNA generated in Example 3-2 were compared with an in silico map indicating the predicted labeling positions when Nb.BssSI was used for nick translation and QD labeling based on the phage λ DNA sequence;

[0099] Figure 23 shows an enlarged image of all the marked sites in the SEM-based DNA optical mapping results of the bacteriophage λ DNA generated in Example 3-2; and

[0100] Figure 24 Shown are the results of SEM-based DNA optical mapping of the genomic DNA derived from HeLa cells generated in Example 3-3. DETAILED DESCRIPTION

[0101] Reference will now be made in detail to the embodiments, examples of which are shown in the accompanying drawings, wherein like reference numerals represent like elements throughout. In this regard, the embodiments may have different forms and should not be construed as limited to the description set forth herein. Accordingly, the embodiments will be described below solely with reference to the accompanying drawings to illustrate various aspects of this specification. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Expressions such as "at least one of...", when preceding a list of elements, modify the entire list of elements, not the individual elements of the list.

[0102] The following examples are provided to further illustrate the present disclosure. However, these examples are for illustrative purposes only and should not be construed as limiting the scope of the present disclosure.

[0103] [Reference example]

[0104] Preparation of DNA-binding proteins

[0105] (1)mNeonGreen-HMG

[0106] The mNeonGreen-HMG plasmid was constructed as described in “21 Fluorescent Protein-Based DNA Staining Dyes. Molecules 2022, 27(16)”.

[0107] The DNA binding motif of the high mobility group (HMG:TPKRPRGRPKK) was fused to the C-terminal region of mNeonGreen (mNG) provided by Dorus Gadella (Addgene plasmid #98882) using polymerase chain reaction (PCR). Complete mNG-HMG was generated by overlap extension PCR. The amplified product was digested with restriction enzymes NdeI (CA-TATG) and BamHI (G-ATCC). The digested product was inserted into the pET15b vector. The amino acid sequence of mNeonGreen-HMG is shown in Table 1 below.

[0108] The constructed mNeonGreen-HMG plasmid was transformed into Escherichia coli BL21 (DE3) strain. BL 21 cells containing the mNG-HMG plasmid were incubated overnight at 37°C in LB medium supplemented with ampicillin. Subsequently, 1 mL of the overnight culture was transferred to 100 mL of LB medium containing ampicillin and incubated at 37°C until the optical density (OD600) at 600 nm reached 0.4-0.6. Protein expression was then induced by adding 1 mM IPTG, followed by incubation overnight at 200 rpm shaking at 20°C to 25°C. After sonication for 15 minutes and centrifugation at 12,000 × g for 10 minutes, mNG-HMG was harvested by affinity chromatography using Ni-NTA agarose resin. mNG-HMG was eluted with a buffer solution containing 50 mM Na2HPO4, 300 mM NaCl and 250 mM imidazole at pH 8.0. The purified protein was buffer exchanged into PBS (phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4), supplemented with glycerol, and stored at -20°C.

[0109] (2) tTALE-emGFP

[0110] The tTALE-emGFP plasmid was constructed as described in “Truncated TALE-FP as DNA Staining Dye in a High-salt Buffer. SciRep-Uk 9, 17197 (2019)”.

[0111] The tTALE-emGFP plasmid was generated by extension PCR by linking the emGFP fluorescent protein to the C-terminal region of the DNA binding protein TALE. The bridging sequence is GGSGG. The constructed tTALE-emGFP plasmid was transformed into the Escherichia coli BL21 (DE3) strain using standard cloning procedures. The amino acid sequence of tTALE-emGFP is shown in Table 1 below.

[0112] (3)H-NS

[0113] As described in Analyst 144, 921-927 (2019), the H-NS plasmid was constructed by amplifying the H-NS gene from H-NS-mCherry. Plasmid pET15b was digested with restriction enzymes NdeI (CA-TATG) and BamHI (G-GATCC). Subsequently, the H-NS gene was inserted and ligated into the plasmid using the AccuRapid cloning kit to generate the H-NS plasmid. The constructed H-NS plasmid was transformed into Escherichia coli BL21 (DE3) strain using standard cloning procedures. The amino acid sequence of H-NS is shown in Table 1 below.

[0114] (4)H-NS-mScarlet

[0115] The H-NS-mScarlet plasmid was constructed as described in “AT-specific DNA visualization revisits the directionality of bacteriophage lambda DNA ejection. Nucleic Acids Research (2023)”.

[0116] The H-NS plasmid was digested with BamHI (GATCC). Subsequently, the amplified mScarlet gene was inserted and ligated into the H-NS plasmid using the AccuRapid cloning kit. The constructed H-NS-mScarlet plasmid was transformed into Escherichia coli BL21(DE3) using standard cloning procedures. The amino acid sequence of H-NS-mScarlet is shown in Table 1 below.

[0117] (5)Cro-mNeonGreen

[0118] The Cro-mNeonGreen plasmid was constructed as described in “Negative nanopore sequencing for mapping biochemical processes on DNA molecules. Chemical Communications 59, 9388-9391 (2023)”.

[0119] The Cro-mNeonGreen plasmid is generated by using a cassette vector system by inserting the DNA binding protein encoding gene Cro into the mNeonGreen plasmid. The mNeonGreen plasmid is generated by inserting the mNeonGreen gene between the NdeI (CA2TATG) and XmaI (C2CCGGG) restriction sites in the pET15b vector. Using phage lambda DNA as a template, the Cro gene is amplified by PCR. Standard cloning procedures are used to transform the Cro-mNeonGreen plasmid constructed into the Escherichia coli BL21 (DE3) strain. The amino acid sequence of Cro-mNeonGreen is shown in Table 1 below.

[0120] [Table 1]

[0121]

[0122] Substrate preparation

[0123] Silicon wafers, silicon wafers with a 30 nm thick SiO2 layer, and platinum-coated silicon wafers were purchased from Wafer Market (Yongin, Korea). Positively charged surface-modified silicon wafers were prepared by applying established methods for glass cover slips to silicon wafers with a SiO2 layer. Specifically, the silicon wafer with the SiO2 layer was placed in a Teflon rack and immersed in a piranha etching solution (30:70 v / v H2O2 / H2SO4) for 3 hours. The wafer was thoroughly rinsed with deionized water, and a pH 7 test paper was used to confirm neutral pH. Subsequently, the wafer was sonicated in deionized water for 30 minutes and rinsed again with deionized water to expose the piranha surface. To prepare a 1.1 mM solution, 150 μL of a 50% methanol solution of Q-siloxane (N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (Gelest)) was added to 250 mL of deionized water. The wafer was incubated in 250 ml of a solution containing trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (1.1 mM) at 65°C while stirring at 100 rpm for 16 hours. Subsequently, the wafer was rinsed three times with 99.9% ethanol and stored in 99.9% ethanol to produce a silicon wafer with a cation-modified surface.

[0124] Preparation of PDMS microfluidic devices

[0125] The PDMS microfluidic device was fabricated as described in “Counting DNA molecules on a microchannel surface for quantitative analysis, Talanta 2023, 252: 123826”.

[0126] The PDMS device was fabricated using standard soft lithography replication techniques. First, the microchannel template was deposited in two layers on a silicon wafer using a repeated photolithography procedure, following the protocol described in the Kayaku Advanced Materials SU-8 2000 datasheet. Subsequently, a 20 μm thick layer of SU-8 2015 photoresist was applied to the silicon wafer using a spin coater (Midas System SPIN-1200D, Daejeon, Korea). The spin-coated wafer was exposed to 350 nm radiation through a mask using an aligner (Midas System MDA-400LJ, Daejeon, Korea). After baking and developing the wafer using SU-8 developer, the second layer was precisely patterned on the developed wafer using a maskless lithography system SmartPrint (SmartForce Technologies, La Tronche, France). Due to SmartPrint's compatibility with g-line photoresists, SU-8 TF 6002 was used. After the template wafer was fabricated, the microchannel template located on the silicon wafer was coated with a mixture of PDMS prepolymer and curing agent (10:1 weight ratio) and incubated at 65°C for 12 hours. After the cured PDMS was peeled off from the wafer, the chamber was created by physically punching the channel into the PDMS. Subsequently, the PDMS microchannel was oxidized using an air plasma generator (Femto Science Cute Basic, Korea) at 100 W for 30 seconds. Finally, the PDMS device was cleaned and stored in deionized water to produce the Figure 2 PDMS microfluidic device (100 μm × 2.4 μm) shown.

[0127] Preparation of DNA samples

[0128] Phage lambda DNA (48.5 kb) and circular M13mp18 DNA (7.2 kb) purchased from New England Biolabs were used. Genomic DNA, mitochondrial DNA, and metaphase chromosomes from HeLa cells were extracted using the following method:

[0129] (1) HeLa cells were cultured in DMEM (high glucose, pyruvate) medium supplemented with 10% FBS (fetal bovine serum) and 1X antibiotic-antimycotic (Gibco). When the HeLa cells reached approximately 80% confluence in the cell culture dish, the culture medium was carefully aspirated using a Pasteur pipette. Subsequently, 5 mL of 1X PBS buffer (phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) was slowly added to the side of the culture dish and aspirated. Next, 2 mL of trypsin-EDTA buffer was added, and the culture dish was manually agitated to ensure coverage of the bottom surface. After incubating the cells for 2 minutes, 8 mL of culture medium was added, and the entire solution (10 mL) was transferred to a 15 mL conical tube and centrifuged at 500 g for 10 minutes. The HeLa cells were then resuspended in 1 mL of 1X PBS buffer and transferred to an Eppendorf tube. After washing twice with 1X PBS buffer, the cells were finally resuspended in 500 μL of 1X PBS buffer.

[0130] (2) Preparation of genomic DNA in agarose gel plugs. The cell suspension was mixed with 2% 1X TE LGT solution to achieve a final LGT concentration of 0.7%, and incubated at 4°C for 30 minutes. The DNA plug was washed in 1X TE buffer for 30 minutes, and this step was repeated 4 times. Proteinase K was added to a final concentration of 2 mg / mL, and the mixture was incubated at 42°C for 4 hours. An equal amount of proteinase K was added again, and then incubated at 42°C overnight. The DNA plug was then washed in 1X TE buffer for 30 minutes, and this step was repeated 4 times. Finally, 380 μL of 1X TE buffer was added, and the DNA plug was melted by incubating at 65°C for 20 minutes.

[0131] (3) Mitochondrial DNA was prepared according to the QIAprep Spin Miniprep Kit protocol (Qiagen). The cell suspension was first centrifuged at 1,000 g for 10 minutes. After discarding the supernatant, the cells were resuspended in 300 μL of P1 buffer. Subsequently, 300 μL of P2 buffer was added, followed by 420 μL of N3 buffer. The tube was then centrifuged at 12,000 g for 10 minutes. The resulting supernatant was transferred to a QIAprep spin column and centrifuged at 12,000 g for 1 minute. The plasmid was eluted with 100 μL of elution buffer. The volume of the elution solution was accurately measured and AMPure XP beads were added at 0.4X the measured volume of the solution. The solution was mixed thoroughly for 5 minutes to ensure that the DNA was bound to the beads. Using a magnetic separation stand, the beads were allowed to adhere to the magnet for 1 minute. The solution was slowly removed from the side opposite the magnet-bound beads and 200 μL of 80% ethanol was added. The stand was gently inverted 5 times and the ethanol was removed. This process was repeated twice. Remove any residual ethanol by air-drying the tube at room temperature for approximately 5 minutes with the cap open. Remove the tube from the stand, add 25 μL of 0.1X TE buffer, and mix thoroughly with the beads. After standing at room temperature for 10 minutes, adhere the beads to a magnet using a magnetic separation stand for 1 minute. Mitochondrial DNA is isolated by transferring the solution to another tube.

[0132] (4) Metaphase chromosomes were prepared as follows: 100 μL of colcemid stock solution was added to DMEM medium containing cultured HeLa cells to a final concentration of 0.01 mg / mL. After incubation overnight in a cell culture incubator, the culture medium was collected in a 15 mL centrifuge tube. Subsequently, 2 mL of trypsin-EDTA buffer was dispensed onto the cells, manually stirred to cover the bottom of the culture dish, and incubated for 2 minutes. Then, 5 mL of culture medium was added, and the entire solution was transferred to the same 15 mL centrifuge tube. After centrifugation at 100 g for 5 minutes, the supernatant was removed, and the cells were resuspended in the remaining culture medium. Slowly, 5 mL of 75 mM KCl solution was added along the wall of the tube, and the cells were incubated in this solution for 15 minutes. Subsequently, 5 mL of freshly prepared fixative (methanol: glacial acetic acid 3:1) was slowly added and mixed by inverting the tube. After centrifugation at 200 g for 5 minutes, the supernatant was removed, and the cells were resuspended in 10 mL of fixative. Finally, the previous step was repeated four times and the cells were resuspended in 200 μL of fixative.

[0133] [Example]

[0134] Example 1-1. DNA SEM Imaging Using DNA Binding Proteins and Polymers (mNG-HMG and PVP)

[0135] 25 pg of bacteriophage λ DNA (48.5 kb, New England Biolabs) was mixed with 792 nM mNeonGreen-high mobility group (mNG-HMG, 31 kDa) and incubated for 10 min. The mixture was then diluted with 1× TE buffer (Tris 10 mM EDTA 1 mM pH 8.0), and an equal volume of 5% polyvinylpyrrolidone (PVP, 40 kDa, Sigma-Aldrich) solution was added and incubated for 5 min to prepare a DNA solution.

[0136] 1 μL of the prepared DNA solution was injected into a polydimethylsiloxane (PDMS) microfluidic device mounted on the surface of a positively charged surface-modified silicon wafer (coated with 1.1 mM Q-siloxane), which guided the solution through microchannels (see Figure 2 , indicated by arrows). Before separating the PDMS microfluidic device from the positively charged surface-modified silicon wafer, the solution within the microchannel was dried for 50 minutes, thereby stretching and immobilizing the DNA onto the positively charged surface-modified silicon wafer. The PDMS microfluidic device was then separated from the silicon wafer surface, and the DNA was imaged using a scanning electron microscope (SEM, JEOL, JSM-7100F) at 15 kV and a magnification of 5,000× to 50,000×. For image processing, the resolution of the DNA images was measured using ImageJ software using the "doFWHM" macro.

[0137] Example 1-2. SEM Imaging of DNA Using DNA Binding Proteins and Polymers (H-NS and PVP)

[0138] SEM DNA images were obtained by the same staining, stretching / fixation, and imaging procedures as in Example 1-1, except that 264 nM histone-like nucleoid structural protein (H-NS, 18 kDa) was used instead of 792 nM mNG-HMG.

[0139] Examples 1-3. DNA SEM imaging using DNA binding proteins and polymers (H-NS-mScarlet and PVP) picture

[0140] SEM DNA images were obtained by the same staining, stretching / fixation, and imaging procedures as in Example 1-1, except that 264 nM histone-like nucleoid structural protein-mScarlet (H-NS-mScarlet, 45 kDa) was used instead of 792 nM mNG-HMG.

[0141] Examples 1-4. SEM Imaging of DNA Using DNA Binding Proteins and Polymers (tTALE-EmGFP and PVP)

[0142] SEM DNA images were obtained by the same staining, stretching / immobilization, and imaging procedures as in Example 1-1, except that 396 nM truncated TALE-EmGFP (tTALE-EmGFP, 65 kDa) was used instead of 792 nM mNG-HMG.

[0143] Example 1-5. SEM imaging of DNA using DNA binding proteins and polymers (Cro-mNeonGreen and PVP) picture

[0144] SEM DNA images were obtained by the same staining, stretching / immobilization, and imaging procedures as in Example 1-1, except that 264 nM Cro-mNeonGreen (37 kDa) was used instead of 792 nM mNG-HMG.

[0145] Example 1-6. SEM Imaging of DNA Using DNA Binding Proteins and Polymers (mNG-HMG and PEOX)

[0146] SEM DNA images were obtained by the same staining, stretching / immobilization, and imaging procedures as in Example 1-1, except that 132 nM mNG-HMG was used instead of 792 nM mNG-HMG, and a 1% poly(2-ethyl-2-oxazoline) (PEOX, 50 kDa, Sigma-Aldrich) aqueous solution was used instead of a 5% PVP aqueous solution.

[0147] Example 1-7. SEM Imaging of DNA Using DNA Binding Proteins and Polymers (mNG-HMG and PANI)

[0148] SEM DNA images were obtained by the same staining, stretching / immobilization, and imaging procedures as in Example 1-1, except that 396 nM mNG-HMG was used instead of 792 nM mNG-HMG, and a 0.1% polyaniline (PANI, 40 kDa, Sigma-Aldrich) aqueous solution was used instead of a 5% PVP aqueous solution.

[0149] Example 2-1. SEM imaging of a circular M13mp18 DNA sample (7.2 kb)

[0150] SEM DNA images were obtained by the same staining, stretching / immobilization, and imaging procedures as in Example 1-1, except that circular M13mp18 DNA (7.2 kb) was used as the DNA sample instead of bacteriophage λ DNA.

[0151] Example 2-2. SEM imaging of HeLa cell-derived genomic DNA samples

[0152] SEM DNA images were obtained by the same staining, stretching / fixation, and imaging procedures as in Example 1-1, except that genomic DNA extracted from HeLa cells according to Reference Example (2) was used as the DNA sample instead of bacteriophage λ DNA.

[0153] Example 2-3. SEM imaging of HeLa cell-derived mitochondrial DNA samples

[0154] SEM DNA images were obtained by the same staining, stretching / fixation, and imaging steps as in Example 1-1, except that mitochondrial DNA extracted from HeLa cells according to Reference Example (3) was used as the DNA sample instead of bacteriophage λ DNA.

[0155] Example 3-1. SEM-based DNA optical mapping via QD labeling

[0156] Nick translation was performed after nickase treatment to label the DNA sample with quantum dots. Specifically, a total of 500 ng of M13mp18 double-stranded DNA was incubated at 16 ° C in NEB buffer 2 for 2 hours, and the NEB buffer 2 had 20 units of Nb.BbvCI (New England Biolabs), 10 units of DNA polymerase I (New England Biolabs) and a nucleotide mixture containing biotin-labeled dUTP (biotin-16-dUTP, Jena Bioscience), dTTP, dATP, dCTP and dGTP. After nick translation, the DNA solution was diluted to a final concentration of 10 ng / μL. The resulting solution was then mixed with 30 nM streptavidin-conjugated quantum dots (Qdot 585, Thermo Fisher Scientific) and incubated at room temperature for 10 minutes. To separate DNA from unbound Qdot 585, a 25 nm mixed cellulose-ester membrane was placed on 100 μL of 1× TE buffer, and the DNA solution was applied to the membrane and allowed to stand and incubate for 30 minutes. The DNA solution was then carefully recovered from the membrane. After labeling, the recovered DNA solution was stained, stretched / immobilized, and imaged in the same manner as in Example 1-1 to generate an optical map using SEM.

[0157] Example 3-2. DNA Optical Mapping of λ Phage DNA Samples Using SEM

[0158] SEM-based optical maps were generated by the same labeling, staining, stretching / immobilization, and imaging procedures as in Example 3-1, except that bacteriophage λ DNA (48.5 kb, New England Biolabs) was used instead of circular M13mp18 DNA as the DNA sample, and Nb.BssSI (New England Biolabs) cleaved at the 5′-C^TCGTG-3′ site was used instead of Nb.BbvCI.

[0159] Example 3-3. SEM-based DNA optical mapping of HeLa cell-derived genomic DNA samples

[0160] SEM-based optical maps were generated by the same labeling, staining, stretching / immobilization, and imaging procedures as in Example 3-1, except that genomic DNA extracted from HeLa cells according to Reference Example (2) was used instead of circular M13mp18 DNA as the DNA sample, and Nb.BssSI (New England Biolabs) cleaved at the 5′-C^TCGTG-3′ site was used instead of Nb.BbvCI.

[0161] [Comparative Example]

[0162] Comparative Example 1-1. SEM Imaging of DNA Using DNA Binding Protein (mNG-HMG)

[0163] SEM DNA images were obtained in the same staining, stretching / immobilization, and imaging procedures as in Example 1-1, except that a DNA solution prepared by mixing 25 pg of bacteriophage λ DNA (48.5 kb, New England Biolabs) with 792 nM mNeonGreen-HMG and incubating for 10 minutes was used.

[0164] Comparative Example 1-2. SEM Imaging of DNA Using Polymer (PVP)

[0165] SEM DNA images were obtained in the same staining, stretching / immobilization, and imaging procedures as in Example 1-1, except that a DNA solution prepared by mixing 25 pg of bacteriophage λ DNA (48.5 kb, New England Biolabs) with a 5% PVP (40 kDa) solution and incubating for 10 minutes was used.

[0166] Comparative Example 1-3. SEM Imaging of DNA Using Polymer (PANI)

[0167] SEM DNA images were obtained with the same staining, stretching / immobilization, and imaging procedures as in Example 1-1, except that a DNA solution prepared by mixing 25 pg of bacteriophage λ DNA (48.5 kb, New England Biolabs) with a 0.1% polyaniline (PANI, 40 kDa) aqueous solution and incubating for 10 minutes was used, and the DNA solution was loaded onto a positively charged surface-modified silicon wafer without using a microfluidic device.

[0168] Experimental Example 1. Evaluation of DNA stainability in SEM using various staining agents

[0169] To determine whether the DNA binding protein-polymer complex can function as a staining agent for visualizing DNA under scanning electron microscopy (SEM), the SEM images of phage λ DNA obtained in Examples 1-1 to 1-7 and Comparative Examples 1-1 to 1-3 were used to compare and evaluate the extent of DNA staining. As an untreated control, a DNA solution prepared by diluting 1 ng of λ phage DNA molecules (48.5 kb) in 1 × TE (Tris 10 mM, EDTA 1 mM, pH 8.0) buffer was stretched / immobilized and imaged in the same manner as in Example 1-1, and the resulting SEM images were used. As a control, imaging results obtained by fluorescence microscopy (FM) were used instead of SEM. Unless otherwise stated below, FM images were acquired by the method described in Lee et al., Nucleic Acids Research, Volume 44, Issue 1 (2016).

[0170] Figure 3 Shown are SEM imaging results of bacteriophage λ DNA molecules obtained by stretching / immobilization and imaging in the same manner as in Example 1-1 without any staining agent treatment (untreated control).

[0171] Figure 4 Shown are the SEM imaging results of bacteriophage lambda DNA molecules stained with mNG-HMG alone, then stretched / immobilized and imaged (Comparative Example 1-1), and the corresponding FM imaging results.

[0172] Figure 5 Shown are the results of SEM imaging of bacteriophage lambda DNA molecules stained with PVP alone, then stretched / immobilized and imaged (Comparative Examples 1-2).

[0173] Figure 6 Shown are the results of SEM imaging of bacteriophage lambda DNA molecules stained with a combination of H-NS and PVP, followed by stretching / immobilization and imaging (Examples 1-2).

[0174] Figure 7 Shown are SEM imaging results of bacteriophage lambda DNA molecules stained using a combination of H-NS-mScarlet and PVP, followed by stretching / immobilization and imaging (Examples 1-3), and corresponding FM imaging results.

[0175] Figure 8 Shown are SEM imaging results of bacteriophage lambda DNA molecules stained with a combination of tTALE-EmGFP and PVP, followed by stretching / immobilization and imaging (Examples 1-4), and corresponding FM imaging results.

[0176] Figure 9 Shown are SEM imaging results of bacteriophage lambda DNA molecules stained with a combination of Cro-mNeonGreen and PVP, followed by stretching / immobilization and imaging (Examples 1-5), and corresponding FM imaging results.

[0177] Figure 10 Shown are the SEM imaging results of bacteriophage λ DNA molecules stained using a combination of mNG-HMG and poly(2-ethyl-2-oxazoline), followed by stretching / immobilization and imaging (Examples 1-6), and the SEM imaging results of bacteriophage λ DNA molecules stained using a combination of mNG-HMG and poly(2-ethyl-2-oxazoline), followed by immobilization on a positively charged surface-modified silicon wafer and imaging without the use of a microfluidic device.

[0178] Figure 11 Shown are SEM imaging results of bacteriophage lambda DNA molecules stained using a combination of mNG-HMG and polyaniline, followed by stretching / immobilization and imaging (Examples 1-7).

[0179] Figure 12 Shown are SEM imaging results of bacteriophage lambda DNA molecules stained with polyaniline alone and then immobilized on a positively charged surface-modified silicon wafer without using a microfluidic device (Comparative Examples 1-3).

[0180] Figure 15 Shown are the results of SEM imaging of bacteriophage lambda DNA molecules stained using a combination of mNG-HMG and PVP, followed by stretching / immobilization and imaging (Example 1-1).

[0181] like Figures 3 to 11 As shown, DNA molecules without any staining agent (untreated control), DNA molecules stained with mNG-HMG alone (Comparative Example 1-1), or DNA molecules stained with PVP alone (Comparative Example 1-2) were difficult to visualize under SEM. In contrast, when a DNA-binding protein or a DNA-binding protein-fluorescent protein (such as H-NS, H-NS-mScarlet, tTALE-EmGFP, or Cro-mNeonGreen) was used in combination with PVP (Examples 1-2 to 1-5), DNA appeared as a dark line under SEM, similar to the results obtained when mNG-HMG and PVP were used (Examples 1-1, Figure 15 ). This indicates that successful SEM staining was achieved. In addition, when other polymers, such as poly (2-ethyl-2-oxazoline) or polyaniline, were used, DNA was also successfully stained (Examples 1-6 to 1-7).

[0182] However, if Figure 12As shown, the images of DNA stained with polyaniline alone (Comparative Examples 1-3) formed large aggregates containing branched DNA. Therefore, although polyaniline can bind to DNA without the assistance of proteins, the image quality is significantly degraded.

[0183] These findings demonstrate that efficient electrostaining of DNA molecules for SEM can be achieved by utilizing DNA-binding proteins in combination with polymers capable of binding to them.

[0184] Experimental Example 2. Evaluation of SEM Image Contrast Depending on Substrate Surface Properties

[0185] To evaluate how the surface properties of the substrate used to immobilize DNA molecules affect the contrast in SEM images, droplets of PVP solution were placed on four different surfaces (a positively charged surface-modified silicon wafer, a silicon wafer with a SiO2 layer, a plain silicon wafer, and a platinum-coated silicon wafer), then dried, and the edge of each droplet was captured by SEM. The results are shown in Figure 13 middle.

[0186] Figure 13 Shown are PVP droplets placed on various functionalized silicon wafer surfaces and visualized via SEM. Specifically, a positively charged surface-modified silicon wafer, a silicon wafer with a SiO2 layer, a flat silicon wafer, and a platinum-coated silicon wafer were used. The dashed line in the platinum-coated wafer image indicates the droplet boundary of the PVP solution.

[0187] like Figure 13 As shown, SEM snapshots of the edges of PVP droplets on silicon wafer surfaces (positively charged surface-modified silicon wafers, silicon wafers with SiO2 layers, or ordinary silicon wafers) reveal clear boundaries between dark PVP areas and adjacent wafer areas. However, on the platinum-coated wafer surface, the droplet boundaries indicated by the dotted lines are blurred and difficult to discern. This suggests that on semiconductor surfaces such as silicon wafers, PVP droplets appear as dark shapes because they interfere with secondary electron emission. In contrast, on fully conductive surfaces, such as platinum-coated wafers, the increased passage and emission of secondary electrons through the surface PVP layer reduces the contrast at the droplet boundaries. Therefore, when using PVP to visualize DNA under SEM, the background contrast can be further enhanced using a semiconductor silicon wafer surface. In addition, because the SiO2 layer can immobilize the DNA backbone when derivatized with quaternary ammonium siloxane, subsequent experiments were performed using a positively charged surface-modified silicon wafer surface.

[0188] Experimental Example 3. Evaluation of DNA Extension with or without Microfluidic Device

[0189] To evaluate the effects of using a microfluidic device to supply DNA to a positively charged surface-modified silicon wafer, a DNA solution containing a dye reagent was introduced into the microfluidic device or simply loaded onto a substrate without such a device, and then SEM images were evaluated. Bacteriophage lambda DNA (48.5 kb, New England Biolabs) was used as the DNA sample.

[0190] Specifically, SEM images were obtained in the same manner as in Example 1-1, except that the DNA solution containing the staining reagent was applied to the positively charged surface-modified silicon wafer and dried, and no microfluidic device was used for the extension / immobilization step. For the control group, imaging results obtained by fluorescence microscopy (FM) were used instead of SEM. Figure 14A and 14B The results are shown in Figure 14A and 14B When a microfluidic device was used, staining, stretching / immobilization, and imaging were performed as in Example 1-1, and the resulting SEM images were shown in FIG. Figure 15 middle.

[0191] Figure 14A and 14B Shown are the SEM imaging results and the corresponding FM imaging results of bacteriophage lambda DNA molecules stained with a combination of mNG-HMG and PVP, then applied and immobilized onto the surface of a positively charged surface-modified silicon wafer and imaged without using a microfluidic device, respectively.

[0192] Figure 15 Shown are the results of SEM imaging of bacteriophage lambda DNA molecules stained using a combination of mNG-HMG and PVP, followed by stretching / immobilization and imaging (Example 1-1).

[0193] like Figure 14A and 14B As shown in , even without using a microfluidic device, SEM images show a more complex DNA fine structure compared to FM images. Figure 14A and 15 As shown in the figure, when the microfluidic device is not used, the DNA is randomly arranged, but when the microfluidic device is used, the DNA appears in a parallel, elongated configuration. This indicates that the DNA is guided and stretched by the microchannels of the microfluidic device and becomes immobilized on the positively charged surface of the SiO2 layer.

[0194] Experimental Example 4. Evaluating Resolution in Stained DNA Images

[0195] To evaluate the resolution of SEM-based DNA imaging using staining reagents according to embodiments of the present disclosure, samples of bacteriophage lambda DNA (48.5 kb) and circular M13mp18 DNA (7.2 kb) were used to compare the thickness of DNA in SEM images and FM images.

[0196] Specifically, SEM DNA images of phage λ DNA (48.5 kb) and circular M13mp18 DNA (7.2 kb) were obtained according to Examples 1-1 and 2-1, respectively. For the control group, DNA thickness was observed in images obtained by fluorescence microscopy (FM) rather than SEM. The results are shown in Figure 16A and 16C In addition, to accurately measure DNA thickness, the “doFWHM” ImageJ macro (downloaded from GitHub) was used to calculate the full width at half maximum (FWHM) of the intensity distribution derived from the inverted SEM DNA image, and Figure 16B The results of these calculations are shown.

[0197] Figure 16A Shown are the SEM imaging results of bacteriophage λ DNA (48.5 kb) stained using a combination of mNG-HMG and PVP, followed by stretching / immobilization and imaging (Example 1-1), and the corresponding FM imaging results. Figure 16B Shown Figure 16A A magnified inverted version of the SEM imaging results of λ DNA (48.5 kb) in , along with the intensity distribution and measured FWHM value along the yellow line of the inverted SEM DNA image.

[0198] Figure 16C Shown are SEM imaging results of circular M13mp18 double-stranded DNA (7.2 kb) stained using a combination of mNG-HMG and PVP, followed by stretching / immobilization and imaging (Example 2-1), and corresponding FM imaging results.

[0199] like Figure 16A As shown, although the length of the λ DNA remains constant in both the SEM and FM images, a significant difference in thickness is observed. Specifically, the SEM image shows finer continuous black lines at higher resolutions compared to the FM image, with respective thickness values ​​of approximately 15 nm versus 450 nm, indicating significant parallax.

[0200] In addition, if Figure 16BAs shown, the FWHM of the SEM DNA images averaged 15.0 ± 4.0 nm (ranging from 9.4 nm to 22.8 nm), while the FWHM of the FM DNA images averaged 239 ± 30 nm. The FWHM measured in the inverted SEM DNA images was 9.4 nm, consistent with observations from the inverted SEM images.

[0201] The FWHM calculations were based on the following assumptions: the DNA molecule has a thickness of 2 nm, the mNeonGreen-HMG protein has a diameter of 2.4 nm and a height of 4.2 nm, and the PVP polymer has a weight average molecular weight (Mw) of 40 kDa and a radius of gyration (Rg) of 3.8 nm, as reported in “Enhancement in Elastic Bending Rigidity of Polymer Loaded Reverse Microemulsion, Langmuir 2017, 33(45):13014-13026”. Assuming that the PVP polymer is annular, its outer diameter (d_out) is approximately 9.8 nm according to the following equation 1. The assembly of proteins and polymers around the DNA molecule may contribute to Figure 16B An average thickness of 15.0 ± 4.0 nm was observed in the PDMS PDMS samples.

[0202] [Equation 1]

[0203]

[0204] In addition, if Figure 16C As shown, high-resolution SEM images vividly capture circular or supercoiled DNA structures, which appear only as small dots in FM images.

[0205] These findings indicate that SEM-based DNA imaging using a staining reagent according to an embodiment provides a resolution of 15 nm, which means that the resolution of SEM is far superior to the approximately 239 nm resolution of DNA images obtained by FM.

[0206] Experimental Example 5. Visualization of Various Types of DNA Molecules

[0207] To determine whether SEM-based DNA imaging using a staining reagent according to an embodiment can visualize DNA molecules derived from human cells, SEM images were evaluated for genomic DNA, mitochondrial DNA, and metaphase chromosomes extracted from HeLa cells.

[0208] Specifically, SEM DNA images of HeLa cell-derived genomic DNA and mitochondrial DNA were obtained according to Examples 2-2 and 2-3, respectively. For the control group, images obtained by FM instead of SEM were used. The results are presented in Figures 17A to 17C as well as Figure 18A and 18B middle.

[0209] In addition, in order to observe metaphase chromosomes extracted from HeLa cells, 2 μL of the cell solution prepared according to Reference Example (4) was placed on a positively charged silicon wafer and dried. To remove the cell membrane, the silicon wafer was first immersed in distilled water for 15 minutes and then treated with 0.15 M phosphate buffer (pH 6) containing 0.0125% trypsin for 1 minute. Next, the wafer was washed with distilled water and incubated in the same phosphate buffer at 37°C for 1 hour. Finally, after further incubation in distilled water for 3 hours, the silicon wafer was immersed in a 5% PVP solution or a YOYO-1 solution. Subsequently, SEM or FM images were obtained. The results are shown in FIG. Figures 19A to 19C middle.

[0210] Figure 17A and Figure 17B The SEM imaging results (Example 2-2) and the corresponding FM imaging results of genomic DNA molecules extracted from HeLa cells, stained using a combination of mNG-HMG and PVP, and then stretched / immobilized and imaged, are shown respectively. Figure 17C Shown are SEM imaging results of Christmas tree-like DNA structures potentially indicating transcription along the backbone of HeLa cell-derived genomic DNA.

[0211] Figure 18A and Figure 18B Shown are the SEM imaging results (Example 2-2) and the corresponding FM imaging results of mitochondrial DNA (16.6 kb) extracted from HeLa cells, stained using a combination of mNG-HMG and PVP, and then stretched / immobilized and imaged.

[0212] Figure 19A Shown are the results of FM imaging after metaphase chromosomes extracted from HeLa cells were immobilized on a positively charged surface-modified silicon wafer and stained with YOYO-1 staining reagent.

[0213] Figure 19B Shown are the results of SEM imaging after metaphase chromosomes extracted from HeLa cells were immobilized on a positively charged surface-modified silicon wafer and then stained with PVP alone in the absence of DNA-binding proteins.

[0214] Figure 19CShown are the results of SEM imaging after immobilizing metaphase chromosomes extracted from HeLa cells on a positively charged surface-modified silicon wafer without staining with any DNA-binding protein or PVP, thereby observing the native state of the chromosomes.

[0215] like Figure 17B As shown in , the FM images appear coarse and occasionally tangled, making it difficult to determine the structure, and the fluorescence intensity of the DNA images exhibits considerable variation. Figure 17A As shown, SEM images clearly reveal the DNA and its spatial distribution. SEM images can identify multiple DNA molecules in close proximity that cannot be distinguished by FM images.

[0216] In addition, if Figure 17C As shown, a "Christmas tree-like" structure indicative of chromatin transcription was identified—which reportedly could not be resolved by FM due to diffraction limits. So far, electron microscopy images depicting such structures have been reported in amphibian oocytes, insects, yeast, and mice, but not in human cells or cell lines. Figure 17C In the figure, yellow arrows presumably indicate the DNA backbone, while the increasingly dense brush lines presumably represent transcribed RNA molecules.

[0217] like Figure 18B As shown in Figure 2, despite being 16.6 kb in length, mitochondrial DNA extracted from HeLa cells appears only as dots in FM images. Figure 18A As shown, SEM images reveal the complex form of circular mitochondrial DNA.

[0218] In addition, if Figure 19A As shown, given that the size of metaphase chromosomes ranges from 5 μm to 10 μm, metaphase chromosomes can be easily observed under FM by staining with YOYO-1. In the case of SEM imaging, attempts to stain metaphase chromosomes with mNG-HMG and PVP resulted in dark cluster images, which is not suitable. Assuming that chromosomes contain abundant proteins, attempts were made to observe metaphase chromosomes by SEM using only PVP without DNA binding proteins. Chromosomes in a natural state without PVP were also observed by SEM. Figure 19B and 19C As shown, SEM can provide high-resolution magnified images of chromosomes even without staining. Thus, metaphase chromosomes can be observed on silicon slides with or without PVP, although chromosomes coated with PVP appear darker due to PVP binding.

[0219] Experimental Example 6. Evaluation of Resolution in DNA Optical Mapping Using SEM

[0220] Based on the confirmation that various types of DNA molecules can be visualized by SEM-based DNA imaging using a staining reagent according to one embodiment, evaluation was performed with various DNA samples to see whether SEM-based optical DNA mapping can exceed the resolution provided by FM-based optical DNA mapping.

[0221] Specifically, as a model system, circular M13mp18 double-stranded DNA and Nb.BbvCI (New England Biolabs) cleaved at the 5'-CC^TCAGC-3' site were used according to the embodiment to generate a DNA optical map based on SEM (Example 3-1). For control, a DNA optical map based on FM obtained by imaging Example 3-1 with FM instead of SEM was used. The results are shown in Figure 21B and 21C middle.

[0222] In addition, as another model system, according to the embodiment, bacteriophage lambda DNA and Nb.BssSI (New England Biolabs) cut at the 5'-C^TCGTG-3' site were used to generate a DNA optical map based on SEM (Example 3-2). For the control, the DNA optical map based on FM obtained by imaging Example 3-2 with FM instead of SEM was used. The results are shown in Figure 20B 、 20C , 22 and 23.

[0223] In addition, as a model system, genomic DNA extracted from HeLa cells and Nb.BssSI (New England Biolabs) cleaved at the 5'-C^TCGTG-3' site were used to generate DNA optical maps based on SEM (Example 3-3). The results are shown in Figure 24 middle.

[0224] Figure 20A is a schematic diagram illustrating DNA stained with mNG-HMG (DBP-FP) and PVP (indicated by black circles) and labeled with streptavidin-coated quantum dots. Figure 20B and 20C An enlarged view of the QD-labeled sites from the SEM-based DNA optical map of the bacteriophage λ DNA generated in Example 3-2 and an enlarged view of the QD-labeled sites from the corresponding FM-based DNA optical map are shown, respectively.

[0225] Figure 21Ais a schematic diagram illustrating that circular M13mp18 double-stranded DNA (7.2 kb) contains two nicking sites at positions 1373 bp and 1417 bp and that DNA polymerase proceeds in two opposite directions. Figure 21B and 21C The results of observing QD labels in the DNA optical map based on SEM and the corresponding QD labels in the DNA optical map based on FM of the circular M13mp18 double-stranded DNA generated in Example 3-1 are shown respectively.

[0226] Figure 22 The SEM-based DNA optical mapping results and the corresponding FM-based DNA optical mapping results of phage λ DNA generated in Example 3-2 were compared with a computer map indicating the predicted labeling positions when Nb.BssSI was used for nick translation and QD labeling based on the phage λ DNA sequence.

[0227] Figure 23 Shown are magnified images of all marked sites from the SEM-based DNA optical mapping results of the bacteriophage λ DNA generated in Example 3-2.

[0228] Figure 24 Shown are the results of SEM-based DNA optical mapping of the genomic DNA derived from HeLa cells generated in Example 3-3.

[0229] like Figure 20B and 20C As shown in Figure 2, dozens of QDs are clearly distinguishable in the SEM image, while those QDs are merged into a single spot in the FM image. Figure 21B and 21C In the figure, the FM image shows the merged signal from the green FP and red QDs as a single bright yellow spot; however, a single SEM image shows 20-30 QD labels clearly distributed along the circular DNA backbone.

[0230] In addition, if Figure 22As shown, the computer map predicted a total of eight marker sites. However, in the FM-based optical map, due to the short distance between some of the marker sites, it was difficult to observe all eight sites - specifically, the fifth and sixth sites were 789bp apart (789bp × 0.34nm / bp = 268nm), and the seventh and eighth sites were 321bp apart (321bp × 0.34nm / bp = 109nm). In the FM-based optical map, the fifth and sixth sites were merged into a single point (789bp, 268nm), and the seventh and eighth sites were also merged into a single point (321bp, 109nm). Therefore, only six red marker sites were distinguished in the FM-based optical map. In contrast, the SEM-based optical map successfully revealed all eight sites, with each site showing five QDs, as shown in the magnified images of the seventh and eighth sites. In addition, as shown in the magnified images of the seventh and eighth sites Figure 23 As shown, QD labeling was confirmed at all eight sites. Since each cluster composed of multiple nanoparticles accurately identified the location of sequence-specific nick translation, these findings indicate that the SEM-based optical DNA mapping of the present disclosure achieves significantly higher resolution than FM-based optical DNA mapping.

[0231] In addition, if Figure 24 As shown, successful visualization of the DNA backbone and QD-labeled regions was achieved using genomic DNA from HeLa cells, demonstrating the ability of the disclosed SEM-based optical DNA mapping to perform high-resolution optical mapping of human genomic DNA.

[0232] According to one aspect, by utilizing SEM as the basic platform, the method of optical DNA mapping offers several advantages: enhanced accessibility and user-friendliness, excellent compatibility with chemically functionalized surfaces and microfluidic devices, and the ability to obtain high-resolution DNA images without the use of metals, thereby enabling optical DNA mapping with improved resolution.

[0233] It should be understood that the embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. The description of features or aspects within each embodiment should generally be considered to be applicable to other similar features or aspects in other embodiments. Although one or more embodiments have been described with reference to the accompanying drawings, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of this disclosure as defined by the appended claims.

Claims

1. A method for optical DNA mapping based on scanning electron microscopy (SEM), the method comprising: (a) contacting the double-stranded DNA with a nicking enzyme to form a nick at the target sequence motif; (b) generating labeled double-stranded DNA by incorporating a labeled nucleotide into the nick site of the nicked double-stranded DNA; (c) staining the backbone of the labeled double-stranded DNA; (d) stretching and immobilizing the dyed double-stranded DNA on a substrate with a functionalized surface; as well as (e) The distance between each marker was determined by imaging the immobilized double-stranded DNA using a scanning electron microscope (SEM).

2. The method of claim 1, wherein the nicking enzyme is Nb.BbvCI, Nb.BssSI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, nCAS9, or a combination thereof.

3. The method of claim 1, wherein the labeled nucleotide is a nucleotide directly or indirectly conjugated to a detectable label. The method of claim 3 , wherein the detectable label is a nanoparticle or a fluorescent dye.

5. The method according to claim 4, wherein the nanoparticles are any one selected from the group consisting of metal nanoparticles, oxide nanoparticles, sulfide nanoparticles, nanoclusters, quantum dots, graphene quantum dots, perovskites, carbon dots, polymer particles, hydroxyapatite and magnetic nanoparticles.

6. The method of claim 1, wherein step (b) comprises: contacting the nicked double-stranded DNA with a DNA polymerase in the presence of at least one nucleotide conjugated to a first binding moiety to generate a modified double-stranded DNA having the first binding moiety; as well as contacting the modified double-stranded DNA with a detectable label conjugated to a second binding moiety to generate labeled double-stranded DNA, The second binding portion forms a binding pair with the first binding portion.

7. The method according to claim 6, wherein the first binding moiety or the second binding moiety is any one selected from the group consisting of biotin, streptavidin, digoxigenin, neutravidin and avidin. The method according to claim 6 , wherein the DNA polymerase has 5′→3′ exonuclease activity.

9. The method of claim 1, wherein step (c) comprises contacting the labeled double-stranded DNA with a DNA-binding protein.

10. The method of claim 9, wherein the DNA binding protein comprises a fluorescent protein.

11. The method according to claim 9, wherein step (c) further comprises contacting the double-stranded DNA with a polymer capable of binding to the DNA-binding protein, an anhydrate thereof, or a salt thereof.

12. The method according to claim 11, wherein the polymer, anhydrate thereof, or salt thereof comprises at least one structural unit represented by Formulae 1 to 9 and has a weight average molecular weight (Mw) of 10 kDa to 100 kDa: [Formula 1] [Formula 2] , , [Formula 3] [Formula 4] [Formula 5] , , , [Formula 6] [Formula 7] , , [Formula 8] , [Formula 9] ， In formulas 1 to 9, m+n is 1.

13. The method of claim 1, wherein step (c) is performed in a metal-free manner.

14. The method according to claim 1, wherein the substrate having a functionalized surface is a silicon substrate that is positively charged by surface modification.

15. The method according to claim 14, wherein the silicon substrate is a silicon substrate having a SiO2 layer formed thereon.

16. The method of claim 14, wherein the surface modification is performed with an ammonium salt.

17. The method of claim 1, wherein step (d) comprises passing the stained double-stranded DNA through a microchannel.

Images